Radiopharmaceuticals with application in diagnostic imaging and therapy have important roles in modern medicine. For the most part, these radioisotopes are radiometals which are either main group metals or part of the lanthanide series. This family of radioisotopes possess a plethora of nuclear and chemical properties, which can be utilised in diagnostic and therapeutic applications. In addition, this family possess a number of advantages over their non-metal counterparts. For instance, radio-labelling using radiometals is typically the final synthetic step and through development and optimisation is usually high-yielding. Furthermore, certain radiometals can be produced conveniently and cost efficiently (within GMP requirements) by means of an on-site generator.
However, in nearly all cases the free (cationic) metal ions are inherently toxic with defined bio-distributions. In order to render these radiometals biologically applicable and useful the metal cations must be complexed by a suitable ligand (organic based chelator). Ligands can be used to ‘shield’ the metal ions from the environment which may otherwise compromise their intended use.
Whilst, the exact nature of the resulting complex (size, redox properties, donor groups, charge, size) will influence its biodistribution, it is widely acknowledged that in order to create a effective, useful and applicable biodistribution it is necessary to incorporate some form of targeting vector with the radio-label. One method for doing so involves the covalent attachment of the targeting vector, in some cases via a coupling unit, to the ligand. Compounds which possess both a chelating and targeting unit are commonly referred to as bifunctional chelators (BFC's).
The development of ligands for the complexation of main group and lanthanide metal ions has been a focus of research for many years, and driven for the most part by advances in medicine. For instance the development of magnetic resonance imaging (MRI) for diagnosis, initiated the development of contrast agents which enhance the image quality. For these applications, paramagnetic complexes of gadolinium in particular have proven effective and are preferred. As a consequence there has been considerable focus on the development of new ligands suitable for in vivo application. Due to the chemical similarity of metals in the lanthanide series, many of these ligands can be used with other lanthanide ions. Of particular interest in this regard are radionuclides such as 153Sm, 177Lu, 166Ho and 90Y (a ‘lanthanide-like’ radiometal), which are potentially useful as radiopharmaceuticals. This similarity within the lanthanide series has resulted in the emergence of a new field entitled THERANOSTICS (THERApy and diagNOSTICS). The premise is that once a ligand has been designed which works effectively as part of a complex in diagnostic imaging it could then, in theory, be applied in therapeutic applications simply be changing the metal radionuclide used.
In terms of metallo-radiopharmaceutical there are two critical pre-requisites which should be met, if they are intended for human use. Firstly, the radio-labelled complex should be stable in vivo over at least the intended application of the radiopharmaceutical. Secondly, due to the inherent time constraints arising from the half-life of the radionuclide of interest, radio-labelling should be efficient and fast. Therefore, the suitability of a BFC is evaluated in terms of its kinetic (and to a lesser extent it's thermodynamic) stability, and rate of radio-labelling. Whilst a high thermodynamic complex stability is desirable for in vivo application such complexes are typically more difficult to form, requiring longer reaction times and greater energy input.
Gallium is a main group metal with three radioisotopes which can be used in radiopharmaceuticals. 66Ga and 68Ga are positron emitters, with half-lives of 9.5 h and 67.7 min respectively, and 67Ga is a γ(gamma)-emitter with a half-life of 3.26 days. 68Ga represents one of the very early radionuclides applied in Positron Emission Tomography (PET). In recent times there has been somewhat of a renaissance of the 68Ga radionuclide, which is largely a result of the availability of the radionuclide from a cost effective and convenient generator. Modern generators are eluted using hydrochloric acid provide “cationic” 68Ga, as opposed to the “inert” 68Ga complexes extracted from previous generations. In addition to this 68Ga is favoured due to its chemical and radiochemical properties, which allow for the formation of sufficiently stable radio-labelled complexes with desirable radiochemical decay characteristics (high β+-emission yield of 89%, suitable γ-ray energy, convenient half-life of 67.7 min).
Largely following the development of MRI and Single Photon Emission Computed Tomography (SPECT) imaging agents, the 68Ga cation was initially chelated using existing ligands. Namely; EDTA-(ethylenediamintetraacetic acid), DTPA-(diethylenetriaminepentacetic acid) or DOTA-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) based derivatives (Figure I). The replacement of 111In-Octreoscan (DTPA with a coupled octreotide targeting vector) by 68Ga-DOTA-octreotides in clinical PET/CT imaging of neuroendocrine tumours, has paved the way not only for clinical acceptance of 68Ga-radiopharmceuticals, but also given recognition to the potential of the 68Ga/68Ga radionuclide generator.

Ligands currently used to chelate 68Ga(III) and provide a means for attachment of a targeting vector are mostly derivatives of DOTA and NOTA (1,4,7-triazacyclononane-1,4,7-tricetic acid: Figure I). Whilst both ligands give rise to 68Ga radio-labelled complexes of exceptionally high stability, they possess inherent disadvantages. DOTA ligand derivatives in particular are not well-suited for 68Ga chelation; a feature which is manifested as the long reaction times and high temperatures required for radio-labelling.
The NOTA ligand derivatives are much better matched to the coordination requirements of 68Ga(III), which largely addresses these problems without detriment to the thermodynamic stability. However, NOTA derivatives are disadvantaged by the acidic pH conditions required for radio-labelling (which limits the biological targeting vectors which can be used), cost and difficult syntheses. Whilst they serve the purpose, improvements to increase efficiency, suitability, application and costs are required to realise the potential of the favourable 68Ga-radionuclide. Specific areas of improvement include quantitative radio-labelling at milder pH and lower temperatures, whilst maintaining sufficient in vivo stability. These improvements are important for the widespread use and development of 68Ga as the radionuclide of choice for PET/CT.
It is interesting to note that, with few exceptions, trends have seen a deviation away from rigid cyclic chelators (DOTA and NOTA) towards more flexible acyclic systems. Acyclic ligands are typically synthetically less challenging and tend to radio-label more rapidly; however the absence of the macrocyclic effect renders them kinetically less stable to metal dissociation.
In the last few years several acyclic and macrocyclic ligands have been reported, which are thought to have favourable properties for the chelation of 68Ga(III) (Figure II).

Ideally ligands should radio-label with 68Ga(III) rapidly (<10 min) at mild pH and temperature. Furthermore they should provide a point/s of attachment of a targeting moiety, which are isolated from the inert chelating core, and be selective for 68Ga(III) over other metal cations such as iron(III).
At present 99mTc radiopharmaceuticals can be prepared using a kit-type radio-labelling protocol, in which the lyophilised ligand (and other compounds required for labelling, such as a buffer) is contained in a vial to which the generator eluate is directly added to initiate the labelling. The 68Ge/68Ga generator has a longer shelf life that the 99Mo/99mTc generator, and therefore the development of a similar kit-type radio-labelling protocol would be a considerable step towards the clinical application and widespread adoption of 68Ga radiopharmaceuticals. This requires the development of BFCs that label quantitatively and rapidly at room temperature, physiological pH, low BFC concentrations and in the presence of competing metal ions.
Ligands featuring the 1,4-diazepine (DAZA: Figure II) scaffold have been investigated in four main areas. These are as:                heptadentate ligands for complexes which may act as diagnostic (MRI and PET) imaging agents        hexadentate ligands for manganese(II) complexes for potential application as contrast agents for MRI        tetra- and penta-dentate ligands for potential application in rare earth metal catalysis        tridentate ligands for transition metals        
Of particular relevance to this work are the complexes relating to imaging applications. There are currently two accepted patents (the second being a continuation of the first) which are related to the invention claimed here. A description encompassing this work, and related literature, is of relevance to this invention.
The compounds patented (U.S. Pat. No. 7,893,223 B2 and U.S. Pat. No. 7,186,400 B2) are defined by the structural formula shown in Figure III. The nature of the variable groups (FG and Y1-5) is broad, and encompasses a wide range of chemical functionalities. Nx designates the exocylcic nitrogen atom.

The variable groups (FG and Y1-5) are defined by the following chemical functionalities:                In the instance of 1,4-diazepine derivatives, the Y1 groups are taken together to form a straight or cyclic C2-C10 alkylene group, or an ortho-disubstituted arylene.        Y2-5 are an H, carboxy, or an optionally substituted group selected from C1-C20 alkyl, C3-C10 cycloalkyl, C4-C20 cycloalkylalkyl, aryl, arylalkyl, a group bearing an acidic moiety and a group bearing an amine moiety, each of which may be further optionally substituted with functional groups selected from the group consisting of carboxyl, amino, aldehydes, haloalkyl, maleimidoalkyl, hydroxyl and sulphydryl groups which allow conjugation with a suitable molecule able to interact with physiological systems.        FG may be the same or different, are carboxy, —PO3H2 or —RP(O)OH, wherein R is hydrogen, or an optionally substituted group selected from C1-C20 alkyl, C3-C10 cycloalkyl, C4-C20 cycloalkylalkyl, aryl, arylalkyl, a group bearing an acidic moiety and a group bearing an amine moiety, each of which may be further optionally substituted with functional groups selected from the group consisting of carboxyl, amino, aldehydes, haloalkyl, maleimidoalkyl, hydroxyl and sulphydryl groups which allow conjugation with a suitable molecule able to interact with physiological systems.        
The vast majority of research which utilisises the 1,4-diazepine scaffold (DAZA: Figure IV), and its derivatives, has focused on the chelation of paramagnetic metal ions and the use of the corresponding complexes as contrast agents for MRI. Complexes aimed at this application have comprised of a common chelating unit in which Y1 is joined to form an ethylene bridge. All FG groups are carboxy, and Y3-5 are hydrogens. The ‘parent’ ligand has Y2 as a methyl group to give 6-amino-6-methzlperhydro-1,4-diazepinetertaacetic acid (AAZTA: Figure IV). The gadolinium(III) AAZTA complex displays favourable characteristics in terms of stability and relaxivity. The favourable relaxivity is consistent with the presence of two inner sphere coordinated water molecules and simultaneous favourable residence times of the coordinated water molecules. Derivatisation of this ligand to yield BFC's has focused exclusively on conjugation via Y2. Some examples for AAZTA derivatives which have been used for the synthesis of BFC's and more complex structures are shown in Figure IV.

There are three examples in the literature of derivatives of the parent AAZTA ligand which included site modification other than Y2. Botta et al investigated the usefulness of derivatives with reduced denticity, by replacing one of the FG groups (carboxy) with a coordinatively inert functioning group (Figure V). The resulting manganese complexes, sought as potential Mn(II) MRI contrast agents, were not sufficiently stable for application in vivo. Similar metal complex stabilities were echoed in the work of Hegetscheweiler which found that the functionalised triamine, 1,4-diazepine (DAZA), scaffold was poorly suited to the complexation of transition metal cations.

Parker and co-workers sought to modify the relaxivity of AAZTA derivatives, by functionalising one of the pendant acetate groups at Y3 by the substitution of a proton for an alkyl carboxyl group (Figure VI). Subsequently, the suitability of the scaffold for use as lanthanide based fluorescent probes was investigated by replacing the endocyclic substituents (R3 and connected FG) with sensitising chromophores (Figure VI). Neither derivatisation produced results which implored further development along these lines.

There is a single example in literature of a 68Ga complex featuring an AAZTA ligand (Figure VII). Binding affinity studies of the targeting vector with its receptor indicated that, AAZTA based BFC had the most detrimental effect on the binding affinity of the targeting vector (in comparison with BFC analogous of DTPA and DOTA analogues). Like all other examples, the targeting vector is attached to the chelating unit via Y2.
